Diagnostic ultrasonic imaging is based on the principle that waves of sound energy can be focused upon an area of interest and reflected in such a way as to produce an image thereof. The ultrasonic transducer is placed on a body surface overlying the area to be imaged, and ultrasonic energy in the form of sound waves is directed toward that area As ultrasonic energy travels through the body, the velocity of the energy and acoustic properties of the body tissue and substances encountered by the energy determine the degree of absorption, scattering, transmission and reflection of the ultrasonic energy. The transducer then detects the amount and characteristics of the reflected ultrasonic energy and translates the data into images.
As ultrasound waves move through one substance to another there is some degree of reflection at the interface. The degree of reflection is related to the acoustic properties of the substances defining the interface. If these acoustic properties differ, such as with liquid-solid or liquid-gas interfaces, the degree of reflection is enhanced. For this reason, gas-containing contrast agents are particularly efficient at reflecting ultrasound waves. Thus, such contrast agents intensify the degree of reflectivity of substances encountered and enhance the definition of ultrasonic images.
Ophir and Parker describe two types of gas-containing imaging agents: (1) free gas bubbles; and (2) encapsulated gas bubbles (Ultrasound in Medicine and Biology 15(4):319-333 (1989)), the latter having been developed in an attempt to overcome instability and toxicity problems encountered using the former. Encapsulated gas bubbles, hereinafter referred to as "microspheres," are composed of a microbubble of gas surrounded by a shell of protein or other biocompatible material. One such imaging agent is ALBUNEX.RTM. (Molecular Biosystems, Inc., San Diego, Calif.) which consists of a suspension of air-filled albumin microspheres. Microspheres are part of the broader category of imaging agents referred to herein as "microparticles," which intends hollow vessels of gas and/or liquid encapsulated by a shell.
Generally, microparticles of a particular gas in the form of protein-shelled microspheres exhibit improved in vivo stability when compared to free bubbles of the same gas. However, most protein-shelled microspheres still have relatively short in vivo half lives which compromise their usefulness as contrast agents. This instability in vivo was thought to result from the collapse or breakdown of the protein shells under pressure resulting in rapid diffusion of the gas from the microspheres. Thus, many recent efforts have centered on improvements to the protein shell as a way of increasing in vivo pressure stability, such as coating the protein shell with surfactants (Giddy, WO 92/05806), binding the protein with a protein-reactive aldehyde (Feinstein et al., U.S. Pat. No. 4,718,433 and U.S. Pat. No. 4,774,958), covalently cross-linking the protein shell (Holmes et al., WO 92/17213) and ionically cross-linking the protein shell (Klaveness et al., WO 95/23615).
Efforts to stabilize microspheres also include the use of gases other than air, since air-filled microspheres have been shown to quickly lose echogenicity when subjected to pressure changes of 150 mm Hg, such as would be encountered during injection and circulation in vivo (N. deJong, et al., Ultrasound Med. Biol. 19:279-288, 1993.) U.S. Pat. No. 5,413,774 indicates that the pressure resistance of microspheres can be improved by using at least a portion of gas that has a S.sub.gas /MW.sub.gas.ltoreq.0.0031, where S.sub.gas is the water solubility of the gas and MW.sub.gas is the average molecular weight of the gas.
PCT Application WO 95/01187, published Jan. 12, 1995, describes pressure-stable microspheres of gas encapsulated by a heat-insolubilized filmogenic protein wherein the encapsulated gas is entirely a water insoluble gas. Among the gases specifically mentioned are the perfluoroalkanes CF.sub.4, C.sub.2 F.sub.6, C.sub.3 F.sub.8, and C.sub.4 F.sub.10. These microspheres are made by subjecting a mixture of an aqueous solution of the protein and the insoluble gas to ultrasonic or mechanical cavitation in the absence of oxygen by sonicating or milling the mixture in a sonicator/mill that is closed to the atmosphere.
Although the encapsulated gas may be an important factor in determining microparticle stability, the composition and flexibility of the microparticle shell is equally important. In addition to its influence on in vivo stability, the shell composition greatly influences the ability of a microparticle to withstand further processing after formation, i.e. "post-processing." This can include routine manufacturing procedures, such as sterilization and packaging, as well as additional chemical manipulations. For example, it may be desirable to perform the chemical steps necessary to attach targeting moieties such as antibodies to the microparticle shell after formation.
It is well known in the art to target therapeutic or diagnostic agents to specific tissue or organs to enhance the efficacy of such agents. Antibodies have been used extensively to target cytotoxic agents or detectable labels to cancers. For instance, radioimmunodetection techniques employing radiolabeled antibodies to various cancer types have been employed to detect and image cancerous tissue. Similarly, toxins such as ricin have been conjugated to antibodies specific to cancer to produce cancer-specific immunotoxins. Antibodies have also been bound to particulate materials such as liposomes and albumin particles. In this regard, U.S. Pat. No. 5,216,130 describes Tc-99m-labeled albumin microspheres conjugated to IgG via dextran spacer arms.
In addition to antibodies, other ligands that are specific to receptors on particular cell types have been used to target agents to such cells. For instance, it is known that hepatocytes possess plasma membrane receptors for glycoproteins whose oligosaccharide chains have .beta.-linked galactose or N-acetylgalactosamine terminals (Ashwell, G. et al. Adv. Enzymol. 41:99 (1974) and Ann. Rev. Biochem. 51:531 (1982)). Liposomes and magnetic particles whose surfaces have been galactosylated or coated with arabinogalactan are rendered hepatocyte-specific. Such magnetic particles have been shown to be useful as MRI agents for imaging the liver (Josephson, L. et al., Mag. Res. Imag. 8:637-646 (1990)). Asialoglycoproteins have also been conjugated to genes to target genes to hepatocytes (PCT/US 92/03639).
Although attaching functional moieties such as antibodies to gas and/or liquid-filled microparticles for use as imaging agents has been previously described (see, for example, PCT Application WO 95/01187), no such protein-shelled microparticles have yet been successfully manufactured for use as in vivo imaging agents. This is partly due to the fragility of the microparticle shells which are incapable of withstanding the physical and chemical conditions which are necessary to make such attachments. Furthermore, such efforts have resulted in shells lacking the ability to recirculate.
It is therefore an object of the present invention to provide non-toxic microparticles with tanned protein shells. These stabilized protein-shelled microparticles are useful as in vivo imaging agents having flexible shells capable of traveling through the capillary system, and which can also serve as intermediates for the production of functionalized microparticles. It is a further object of the present invention to provide processes for the formation of these stabilized microparticles, and processes for the formation of microparticles having functional moieties attached thereto utilizing these stabilized microparticles.